title: Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells.
abstract: BACKGROUND
  PrestoBlue (PB) is a new, simple and extremely fast live assay to monitor cell viability and cytotoxicity. Herein, we compared two in vitro cytotoxicity assays, new (PB) and classic (MTT), in the assessment of viability of human umbilical vein endothelial cells (HUVECs) in the presence of selected plant extracts.
  
  
  METHODS
  The anti-proliferative effects of two extracts from medicinal plants, i.e., walnut husk extract and spent hop extract, used at the concentration range of 1-200 μg/ml of gallic acid equivalent, were compared with the effects recorded for resveratrol--a natural polyphenolic compound. Reduction of dyes by endothelial cells was determined colorimetrically (MTT and PB) and fluorometrically (PB).
  
  
  RESULTS
  At higher concentrations, all tested compounds caused significant loss of cell viability. Regardless of plant compound, the PB assay, when measured colorimetrically, produced higher EC50 values compared to other modes of measurement, however, the statistically significant differences in EC50 values among the assays were revealed only for spent hop extract. Conversely, the EC₅₀ values for each plant compound obtained in MTT (colorimetric assay) and PB (fluorometric assay) were similar. According to EC₅₀ values, the cytotoxicity of plant compounds ranked as follows: spent hop extract>resveratrol>walnut husk extract. Furthermore, the MTT assay showed overall lower inter-assay variability and higher signal-to-noise ratio compared to PB assay.
  
  
  CONCLUSIONS
  In conclusion, we recommend fluorometric PrestoBlue assay for cytotoxicity assessment in human endothelial cells. Due to substantial differences in EC₅₀ values and S/N ratios between spectrophotometric PB and MTT or fluorometric PB assays, colorimetric quantification of HUVECs' viability with the use of PB reagent should be avoided.
authors.affiliation: Department of Haemostatic Disorders, Medical University of Lodz, Lodz, Poland. Electronic address: magdalena.boncler@umed.lodz.pl.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Boncler
authors.affiliation: Department of Pharmaceutical Biochemistry, Medical University of Lodz, Lodz, Poland.
authors.forename: Marek
authors.initials: M
authors.lastname: Różalski
authors.affiliation: Department of Pharmaceutical Biochemistry, Medical University of Lodz, Lodz, Poland.
authors.forename: Urszula
authors.initials: U
authors.lastname: Krajewska
authors.affiliation: Department of Biotechnology and Food Sciences, Technical University of Lodz, Lodz, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Podsędek
authors.affiliation: Department of Haemostatic Disorders, Medical University of Lodz, Lodz, Poland.
authors.forename: Cezary
authors.initials: C
authors.lastname: Watala
mesh_terms: D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003124:Colorimetry / Q000379:methods*; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects*; D005470:Fluorometry / Q000379:methods*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D010936:Plant Extracts / Q000494:pharmacology*; D010946:Plants, Medicinal; D000077185:Resveratrol; D059629:Signal-To-Noise Ratio; D013267:Stilbenes / Q000494:pharmacology
chemical_list: D010936:Plant Extracts; D013267:Stilbenes; D000077185:Resveratrol
keywords: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; AB; AlamarBlue; DMF; DMSO; GA; HAECs; HPAECs; HUVEC; HUVECs; MTT; MTT assay; PB; PBS; PrestoBlue; PrestoBlue assay; Resveratrol; SDS; Spent hop extract; Walnut husk extract; dimethyl sulfoxide; dimethylformamide; gallic acid; human aortic endothelial cells; human pulmonary artery endothelial cells; human umbilical vein endothelial cells; phosphate-buffered saline; sodium dodecyl sulphate